猪IgG Fc受体与恒定链的细胞定位及相互关系研究
发布时间：2018-09-09 15:27
【摘要】：克隆猪IgG Fc受体基因(FcRn)及其剪接体基因,研究其与猪恒定链(Ii)的细胞定位及相互关系。利用Triozl法从猪十二指肠组织提取总RNA,进行RT-PCR扩增,PCR产物与pMD-18T载体连接,筛选阳性克隆并序列测定和分析。进一步构建了融合表达绿色荧光蛋白的FcRn基因及其剪接体真核表达载体,利用脂质体Lipofectamine2000介导法与能表达红色荧光的Ii真核表达载体共转染COS-7细胞,共聚焦荧光显微镜检测FcRn基因与猪Ii链在细胞中的共定位,并通过免疫共沉淀进一步研究它们之间的相互关系。序列分析结果表明,FcRn基因序列大小为1 071bp,剪接体片段大小为795bp,两者氨基酸序列均含有胞外结构域、跨膜区和胞浆尾区。细胞定位研究显示,LFC-GFP或SFC-GFP与RFP-Ii共转染COS-7细胞后共定位在细胞的内膜系统。免疫共沉淀结果显示,GFP-Ii与LFC-GFP或SFC-GFP共转染后,检测到LFC-GFP或SFC-GFP的表达。猪Ii链与FcRn基因或剪接体能够形成复合体,共定位于细胞的内膜系统。
[Abstract]:The porcine IgG Fc receptor gene (FcRn) and its splice gene were cloned and their cellular localization and relationship with porcine (Ii) were studied. Total RNA, was extracted from pig duodenum by Triozl method. The product of RT-PCR amplification was ligated with pMD-18T vector. The positive clones were screened and sequenced. Furthermore, the eukaryotic expression vector of FcRn gene and splice was constructed. The eukaryotic expression vector was co-transfected into COS-7 cells by liposome Lipofectamine2000 mediated and Ii eukaryotic expression vector expressing red fluorescence. The co-localization of FcRn gene and porcine Ii chain in cells was detected by confocal fluorescence microscopy, and the relationship between them was further studied by immunoprecipitation. The results of sequence analysis showed that the size of FcRn gene was 1 071bpand the splice fragment was 795bp. both amino acid sequences contained extracellular domain, transmembrane region and cytoplasmic tail region. The study of cell localization showed that LFC-GFP or SFC-GFP co-transfected COS-7 cells with RFP-Ii and then co-located in the intimal system of the cells. The results of co-immunoprecipitation showed that the expression of LFC-GFP or SFC-GFP was detected after co-transfection of GFP-Ii with LFC-GFP or SFC-GFP. Porcine Ii strand and FcRn gene or splice can form a complex, which is located in the endomembrane system of the cell.
【作者单位】： 安徽农业大学动物科技学院;
【基金】：安徽省高校自然科学研究重点资助项目(KJ2015A078) 国家自然科学基金资助项目(30671537)
【分类号】：S828
本文编号：2232773
[Abstract]:The porcine IgG Fc receptor gene (FcRn) and its splice gene were cloned and their cellular localization and relationship with porcine (Ii) were studied. Total RNA, was extracted from pig duodenum by Triozl method. The product of RT-PCR amplification was ligated with pMD-18T vector. The positive clones were screened and sequenced. Furthermore, the eukaryotic expression vector of FcRn gene and splice was constructed. The eukaryotic expression vector was co-transfected into COS-7 cells by liposome Lipofectamine2000 mediated and Ii eukaryotic expression vector expressing red fluorescence. The co-localization of FcRn gene and porcine Ii chain in cells was detected by confocal fluorescence microscopy, and the relationship between them was further studied by immunoprecipitation. The results of sequence analysis showed that the size of FcRn gene was 1 071bpand the splice fragment was 795bp. both amino acid sequences contained extracellular domain, transmembrane region and cytoplasmic tail region. The study of cell localization showed that LFC-GFP or SFC-GFP co-transfected COS-7 cells with RFP-Ii and then co-located in the intimal system of the cells. The results of co-immunoprecipitation showed that the expression of LFC-GFP or SFC-GFP was detected after co-transfection of GFP-Ii with LFC-GFP or SFC-GFP. Porcine Ii strand and FcRn gene or splice can form a complex, which is located in the endomembrane system of the cell.
【作者单位】： 安徽农业大学动物科技学院;
【基金】：安徽省高校自然科学研究重点资助项目(KJ2015A078) 国家自然科学基金资助项目(30671537)
【分类号】：S828
【相似文献】
相关硕士学位论文 前1条
1 万博;猪繁殖与呼吸综合征病毒感染后免疫抑制与IgG Fc受体转录[D];河南农业大学;2010年
，本文编号：2232773
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2232773.html
